We are interested in understanding how normal intestinal cells regulate their growth and how loss of that regulation results in malignant transformation. Our research focuses on molecular mechanisms by which the Src tyrosine kinases and their inhibitors contribute to the regulation. We discovered two fundamental mechanisms whereby Src activity is regulated. One is by addition of a phosphate to a highly conserved tyrosine in the C-terminal tail. Mutation of this site converts the normal cellular Src into a transforming protein. The other is by interaction with Rack1, an endogenous substrate and inhibitor of Src kinases and colonic cell growth. Our recent in vitro studies demonstrate that Rack1 regulates colonic cell growth by suppressing Src activity at critical cell cycle checkpoints and during apoptosis. We hypothesize that Rack1 also regulates cell growth in vivo and plays a key role in mitochondrial apoptosis. To test these hypotheses we will: Aim 1: Analyze mechanisms by which Rack1 regulates growth of intestinal epithelia in vivo. A mouse model with Rack1 deleted in intestinal epithelia will be generated to do so. Rack1's influence on proliferation of crypt and stem cells, migration, differentiation and apoptosis of epithelial cells gene expression and development of intestinal neoplasia will be assessed. We will determine whether loss of Src rescues Rack1 deficiency in intestinal epithelia. Aim 2: Further analyze Rack1 function in mitochondrial cell death. Studies focus on mechanisms by which Rack1 engages and activates proapoptotic Bax and Bim, induces their translocation to mitochondria, mediates their interaction with each other and with prosurvival Bcl-2 family members, regulates Bax sequestration at mitochondria and facilitates Bax oligomerization. Endogenous inhibitors of oncogenic kinases that work at key checkpoints in the cell cycle, in proliferating crypt and stem cells and during apoptosis would exert powerful and pervasive control over cell growth. Exploitation of these multiple functions could be used to develop new and more powerful and selective strategies for treatment of human colon cancer. PUBLIC HEALTH RELEVANCE: The relevance of this proposal to public health lies in discovery of a fundamental mechanism by which the growth of intestinal cells is regulated; by a protein, Rack1, which inhibits a family of cancer-causing enzymes at key checkpoints in the cell cycle (before cells are fated to divide), in proliferating crypt cells and during programmed cell death. Such inhibitors yield powerful and pervasive control over cell growth; they are tumor suppressors and represent exciting new targets for colon cancer therapy.